1. Field of the Invention
This invention relates to a digital microscope.
2. Description of the Related Art
(Note: This application references a number of different publications as indicated throughout the specification by one or more reference numbers within brackets, e.g., [x]. A list of these different publications ordered according to these reference numbers can be found below in the section entitled “References.” Each of these publications is incorporated by reference herein.)
Bacterial motility plays a role in critical environmental and physiological processes, including nutrient cycling, biofouling, and virulence. Despite its importance, motility has only been studied in a few test organisms because of the difficulties of imaging moving micrometer-sized cells. However, the study of the motility of microorganisms is a field which promises to be revolutionized by digital holographic microscopy (DHM) [1]. Because this imaging technique instantaneously probes a large sample volume (milliliters) in three dimensions, it enables the reconstruction of swimming trajectories of essentially unconstrained cells. The feasibility of this approach has been demonstrated in the open ocean for measurements of the distribution and swimming patterns of plankton [2], and investigation of dinoflagellate feeding behavior [3,4]. It has also been used in the laboratory to study motility of algal zoospores [5] and cultured cells [6-12]. However, because of the technical limitations of existing fieldable DHM instruments, such field experiments have so far been restricted to eukaryotic cells>10 μm in diameter. In order to capture bacterial motility, real-time imaging with spatial resolution of <1 μm in all dimensions is required. Such an instrument would allow for in situ investigations of bacterial motility in bodies of water, which has relevance to basic physics and microbiology [13-16] as well as to applications such as water-quality monitoring and astrobiology [17].
Imaging moving bacteria is challenging because of the small size of the cells, their rapid motion (tens to hundreds of cell lengths per second), and their low contrast. Light microscopy relies upon a wide selection of dyes for increasing contrast of specific cell types and subcellular structures. An “off-axis” DHM provides both amplitude and phase images, where contrast in the amplitude image is provided by sample absorptivity at the probe wavelength, and contrast in the phase image results from a difference in index of refraction between the sample and its surrounding medium. Depending upon the organism, one or the other of these image types (or their derived constructs such as phase contrast or DIC) may provide sufficient contrast for identifying and tracking single cells, making dyes unnecessary. This is a distinct advantage over single-beam or “in-line” instruments, where the amplitude and phase images cannot be readily deconvolved without modifications that preclude real-time observation.
In digital holography, recording of the optical interference—fringes—is not done with photographic plates, but with an array detector [18-20]. To record fringes of high contrast over the detector integration time requires: 1) the optical path length difference between the reference arm and science arm be well within the coherence length of the source, 2) that the fringes not shift significantly during the exposure time (which is equivalent to saying the path length variations must be stable to much less than a wavelength at this timescale). For off-axis holography the fringe carrier frequency must also be well sampled by the CCD, in order to accommodate the sample bandwidth [21].
Like classic holography, digital holography enables the reconstruction of an electric field at a given plane a-posteriori—but it relies upon a computer to perform a numerical reconstruction [22-24]. However, it adds two unique capabilities: the ability to numerically reconstruct this electric field at any other plane along the optical axis, and do so as a function of time. In this way, data acquisition consists of a time series of recorded holograms, and afterwards the electric field in a volume is numerically reconstructed for each time stamp, creating a time-lapse movie of a three-dimensional volume.
Several optical configurations have been proposed for the recording step in digital holography: 1) lens-less “Gabor-type” configuration with a simple pinhole divergent illumination [25] or 2) inline holography schemes usually enable compact and straightforward implementation. Although a dual-beam in-line geometry is compatible with phase-shifting [26] (at the price of real-time capability), the previously-mentioned schemes generally cannot discard or deconvolve the contribution from the “twin image” (complex conjugate) of the reconstructed field, hence superposing the final image with an out-of-focus “ghost”. Alternatively, off-axis implementations, using a tilted reference wave to encode the sample wavefront with a fringe pattern, have been employed [18,20]: these provide a spatial multiplexing in the Fourier domain, thus enabling spatial filtering [21] and retrieval of the object complex wavefront free of artifacts. However, off-axis layouts frequently result in rather large instruments, which are alignment-sensitive (notably for accurately dialing the fringe carrier frequency). They are generally less-suited for extreme environments in terms of mechanical and thermal stress with their two-beam geometry (usually Mach-Zehnder or Michelson-type). One or more embodiments of the present invention described a new design that maintains the off-axis implementation, but with a robust optical design which maintains performance.